OCTS technology-based pancreatic cancer and malignant mesothelioma CAR-T therapeutic vector, construction method therefor, and use thereof

ABSTRACT

An OCTS-based CAR-T vector for treating pancreatic cancer and malignant mesothelioma includes lentiviral skeleton plasmid, human EF1α promoter (SEQ ID NO: 14), OCTS chimeric receptor structural domain, and PDL1 single-chain antibody; the OCTS chimeric receptor structural domain consists of CD8 leader chimeric receptor signal peptide (SEQ ID NO: 15), PDL1 single-chain antibody light chain VL (SEQ ID NO: 16), PDL1 single-chain antibody heavy chain VH (SEQ ID NO: 17), mesothelin single-chain antibody light chain VL (SEQ ID NO: 18), mesothelin single-chain antibody heavy chain VH (SEQ ID NO: 19), antibody Inner-Linker (SEQ ID NO: 20), single-chain antibody Inter-Linker (SEQ ID NO: 21), CD8 Hinge chimeric receptor linker (SEQ ID NO: 22), CD8 Transmembrane chimeric receptor transmembrane domain (SEQ ID NO: 23), TCR chimeric receptor T cell activation domain (SEQ ID NO: 26) and chimeric receptor co-stimulator domain.

CROSS REFERENCE TO THE RELATED APPLICATIONS

This application is the national phase entry of InternationalApplication No. PCT/CN2017/110673, filed on Nov. 13, 2017, which isbased upon and claims priority to Chinese Patent Application No.201710390649.2, filed on May 27, 2017, the entire contents of which areincorporated herein by reference.

TECHNICAL FIELD

This invention belongs to the field medical biology, specificallyrelating to a vector, especially an OCTS-based CAR-T vector for treatingpancreatic cancer and malignant mesothelioma. Also, this inventionrelates to the preparation method and application of the vector.

BACKGROUND

The theoretical basis of tumor immunotherapy is that the immune systemcan identify tumor-associated antigens and regulate the body to attacktumor cells (highly specific cytolysis). In the 1950s, Burnet and Thomasmade the theory of “immunological surveillance” that holds thatmutational tumor cells that often occur in the body can be identifiedand eliminated by the immune system, laying a theoretical foundation fortumor immunotherapy [Burnet F M. Immunological aspects of malignantdisease. Lancet, 1967; 1: 1171-4]. Then, a host of tumorimmunotherapies, including cytokine therapy, monoclonal antibodytherapy, adoptive immunotherapy and vaccine therapy, have been appliedto clinical practice.

In 2013, CAR-T, a more advanced tumor immunotherapy, was successfullyput to clinical use, and showed unprecedented clinical effects. CAR-T,short for Chimeric Antigen Receptor T-Cell Immunotherapy, feeds thechimeric molecule composed of promoter, antigen recognition domain,co-stimulator, effector domain, etc. into T cell genome, with transgenicmethod, so as to enable T cells to integrate the recognition, signaltransduction, killing and other functions of target cells to achievespecific killing of target cells [Eleanor J. Cheadle, et al. CAR Tcells: driving the road from the laboratory to the clinic. ImmunologicalReviews 2014. Vol. 257: 91-106]. Clinically, the most leading CAR-T isNovartis' CLT019. For patients having refractory-relapsed acutelymphoblastic leukemia and treated with CLT019, the six-month tumorprogression-free survival rate can reach 67%, and the longest responsetime can be more than two years. By cooperating with hospitals, ShanghaiUnicar Biomedical Technology Co., Ltd., a Shanghai-based company,treated 36 patients with refractory-relapsed acute lymphoblasticleukemia by February 2017, among whom 24 as a percentage of 66.6%experienced complete remission. It is a subversive breakthrough inanti-cancer research. CAR-T may be one of the therapies that are themost likely to cure cancer, and was named the best in top 10breakthroughs of science and technology 2013 by the journal Science.

In spite of current significant effect of CAR-T in treating Blymphocytic leukemia and several other kinds of hematologicmalignancies, there are still some limitations of the therapy. Atpresent, a chimeric antigen receptor (CAR) can only recognize a kind ofantigen target, while tumor cells are a complex colony, and once tumorcells containing corresponding antigens are eliminated, there will berapid proliferation of tumor cells containing no corresponding antigens,thereby giving rise to tumor recurrence after some time. Hence, toenable CAR-T to simultaneously recognize two kinds of antigens, thereare two alternative schemes: one is to transduce two groups of CARs intoprimary T lymphocytes once by building them into a lentiviral transgenicvector; the other is to separately transduce two groups of CARs intoprimary T lymphocytes by transducing two lentiviral transgenic vectorstwice.

The disadvantage of the first scheme is that precious volume oflentiviral transgenic vector is occupied, which is not good for loadingother functional components; efficiency in package of transgenic vectoris low; efficiency in gene transduction is very low, making thetransduction into primary T lymphocytes very difficult.

The disadvantage of the second scheme is that it needs two transductionswith relatively low comprehensive efficiency and long time oftransduction, and primary cells are easy to age, thus resulting indecline in proliferation ability and killing function and affecting theefficacy of tumor clearance.

Mesothelin is a tumor-associated antigen expressed in most malignantpleural mesothelioma, pancreatic cancer, ovarian cancer and some lungcancers and in normal tissues in a narrow range and low quantity, andthus can be used as the immunotherapeutic target for malignant pleuralmesothelioma, pancreatic cancer, ovarian cancer (Gregory L. Beatty, CarlH. June et al. Mesothelin-Specific Chimeric Antigen ReceptormRNA-Engineered T Cells Induce Antitumor Activity in Solid Malignancies.Cancer Immunol Res 2014:2:112-120).

There is the overexpression of PDL1 in most cancerous tissues, includingNSCLC (Non-Small Cell Lung Cancer), melanoma, breast cancer, glioma,lymphoma, leukemia, as well as various urinary tumors, digestive tracttumors and reproductive system tumors [Intlekofer A M, Thompson C B. Atthe bench: preclinical rationale for CTLA-4 and PD-1 blockade as cancerimmunotherapy[J]. J Leukoc Biol, 2013, 94(1):25-39.]. In mouse and humantumor cells, Parsa discovered IFN-γ abnormally secreted by T cells, andIFN-γ can induce the high expression of PDL1 on tumor cells [Ding H, WuX, Wu J, et al. Delivering PD-1 inhibitory signal concomitant withblocking ICOS co-stimulation suppresses lupus-like syndrome inautoimmune BXSB mice[J]. Clin Immunol, 2006, 118(2/3):258-267.]. Highexpression of PDL1 can inhibit the signals of RAS and PI3K/AKT tocontrol the expression of cell cycle checkpoint protein and proteinsrelated to cell proliferation to finally cause the proliferation of Tcells to be suppressed. Through the in-vitro experiments by and mousemodels of Dong, et al, it is also discovered that activation ofPD-1/PDL1 signals can induce the apoptosis of specific CTL, decreasingsensitivity to the cytotoxic effect of CTL and causing the immuneevasion of tumor cells [Dong H, Strome S E, Salomao D R, et al.Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanismof immune evasion [J]. Nat Med, 2002, 8(8):793-800.].

So far, there has been no report on CAR-T overcoming the aforesaiddisadvantages and against the antigens mesothelin and PDL1.

SUMMARY

One of the technical problems to be solved by the invention is toprovide an OCTS-based CAR-T vector for treating pancreatic cancer andmalignant mesothelioma. First of all, it only needs one transductionwith high efficiency in transduction and without prejudice to thecurative effect of CAR-T; second, it doesn't occupy the precious volumeof lentiviral transgenic vector, which is good for loading otherfunctional components; third, it can efficiently close PDL1, blocknegative control signals and be clinically used to suppress tumor immuneevasion, thus improving the curative effect of CAR-T in cellularimmunotherapy.

The second technical problem to be solved by the invention is to providea preparation method of the vector.

The third technical problem to be solved by the invention is to providethe application of the vector.

To solve such technical problems, the invention adopts the followingtechnical scheme:

In the first aspect, the invention provides an OCTS-based CAR-T vectorfor treating pancreatic cancer and malignant mesothelioma, consisting oflentiviral skeleton plasmid, human EF1α promoter, OCTS chimeric receptorstructural domain, and PDL1 single-chain antibody;

The said lentiviral skeleton plasmid consists of AmpR sequencecontaining ampicillin resistance gene and for vast expansion of targetbacterial strain, as shown in SEQ ID NO: 1;

Prokaryotic replicon pUC Ori sequence for plasmid replication, as shownin SEQ ID NO: 2;

Viral replicator SV40 Ori sequence for enhancing replication ineukaryotic cells, as shown in SEQ ID NO: 3; lentiviral packagingcis-element for lentiviral packaging; ZsGreen1 green fluorescentprotein, as shown in SEQ ID NO: 11; IRES ribosome binding sequence, asshown in SEQ ID NO: 12; eWPRE enhanced marmot hepatitis B viruspost-transcriptional controlling element for enhancing transgenicexpression efficiency, as shown in SEQ ID NO: 13;

The sequence of the said human EF1α promoter is shown in SEQ ID NO: 14;

The said OCTS chimeric receptor structural domain consists of CD8 leaderchimeric receptor signal peptide, as shown in SEQ ID NO: 15, PDL1single-chain antibody light chain VL, as show in SEQ ID NO: 16, PDL1single-chain antibody heavy chain VH, as show in SEQ ID NO: 17,mesothelin single-chain antibody light chain VL, as show in SEQ ID NO:18, mesothelin single-chain antibody heavy chain VH, as show in SEQ IDNO: 19, antibody Inner-Linker, as shown in SEQ ID NO: 20, single-chainantibody Inter-Linker, as shown in SEQ ID NO: 21, CD8 Hinge chimericreceptor linker, as shown in SEQ ID NO: 22, CD8 Transmembrane chimericreceptor transmembrane domain, as shown in SEQ ID NO: 23, TCR chimericreceptor T cell activation domain and chimeric receptor co-stimulatordomain, as shown in SEQ ID NO: 26; the said chimeric receptorco-stimulator domains are selected from any one or more combinations of4-1BB, ICOS, CD27, OX40, CD28, MYD88, IL1R1, CD70, TNFRSF19L, TNFRSF27,TNFRSF1OD, TNFRSF13B, TNFRSF18, CD134 and other tumor necrosis factorreceptor superfamilies (TNFRSF).

The said lentiviral packaging cis-element can employ either thesecond-generation lentiviral vector or the third-generation lentiviralvector. The second-generation lentiviral vectors employed by the saidlentiviral packaging cis-element include lentiviral 5 terminal LTR, asshown in SEQ ID NO: 5, lentiviral 3 terminal Self-Inactivating LTR, asshown in SEQ ID NO: 6, Gag cis-element, as shown in SEQ ID NO: 7, RREcis-element, as shown in SEQ ID NO: 8, env cis-element, as shown in SEQID NO: 9, cPPT cis-element as shown in SEQ ID NO: 10. Thethird-generation lentiviral vectors employed by the said lentiviralpackaging cis-element include lentiviral 5 terminal LTR, as shown in SEQID NO: 5, lentiviral 3 terminal self-Inactivating LTR, as shown in SEQID NO: 6, Gag cis-element, as shown in SEQ ID NO: 7, RRE cis-element, asshown in SEQ ID NO: 8, env cis-element, as shown in SEQ ID NO: 9, cPPTcis-element, as shown in SEQ ID NO: 10, and RSV promoter as shown, inSEQ ID NO: 4. The invention preferably employs the third-generationlentiviral vectors.

Preferably, the said PDL1 single-chain antibody light chain VL, as showin SEQ ID NO: 16, PDL1 single-chain antibody heavy chain VH, as show inSEQ ID NO: 17, mesothelin single-chain antibody light chain VL, as showin SEQ ID NO: 18, mesothelin single-chain antibody heavy chain VH, asshow in SEQ ID NO: 19, antibody Inner-Linker, as shown in SEQ ID NO: 20,single-chain antibody Inter-Linker, as shown in SEQ ID NO: 21, areconnected in serial or turned way; specifically, the said serialconnection is that PDL1 single-chain antibody light chain VL isconnected with mesothelin single-chain antibody light chain VL throughsingle-chain antibody Inter-Linker and with PDL1 single-chain antibodyheavy chain VH through antibody Inner-Linker, while mesothelinsingle-chain antibody light chain VL is connected with mesothelinsingle-chain antibody heavy chain VH through antibody Inner-Linker,i.e., pOCTS-PMs (see FIGS. 4A and 4C); specifically, the said turnedconnection is that mesothelin single-chain antibody light chain VL isconnected with mesothelin single-chain antibody heavy chain VH throughantibody Inner-Linker, while PDL1 single-chain antibody light chain VLis connected with mesothelin single-chain antibody heavy chain VHthrough single-chain antibody Inter-Linker, and PDL1 single-chainantibody heavy chain VH is connected with mesothelin single-chainantibody light chain VL through antibody Inter-Linker, i.e., pOCTS-PMt(see FIGS. 4B and 4C).

Preferably, the sequence of the said PDL1 single-chain antibody is shownin SEQ ID NO: 27.

Preferably, there are 6 enhanced nucleotide mutations of the said eWPREenhanced marmot hepatitis B virus post-transcriptional controllingelement: g.396G>A, g.397C>T, g.398T>C, g.399G>A, g.400A>T, g.411A>T.

Preferably, the whole OCTS structural gene expression is started by thesaid human EF1α promoter, and the said CD8 leader chimeric receptorsignal peptide on the N terminal of OCTS coding sequence is used toguide the location of OCTS protein on cytomembrane; the said two groupsof single-chain antibodies PDL1 single-chain antibody light chain VL,PDL1 single-chain antibody heavy chain VH, mesothelin single-chainantibody light chain VL and mesothelin single-chain antibody heavy chainVH are combined into double antigen recognition domain and used torecognize corresponding target antigens; the said CD8 Hinge chimericreceptor linker is used to anchor scFv on the outside of cytomembrane;the said CD8 Transmembrane chimeric receptor transmembrane domain isused to fix entire chimeric receptors on cytomembrane; the said CD28chimeric receptor co-stimulator is used to stimulate in-vitro Tlymphocyte activation and killing effect on in-vivo tumor cells; thesaid CD134 chimeric receptor co-stimulator is used to facilitate Tlymphocyte proliferation and factor secretion and enhance tumorimmunity, which is good for the long-term survival of memory T cells;the said TCR chimeric receptor T cell activation domain is used toactivate the expression of downstream signals; the said PDL1single-chain antibody can efficiently close PDL1, block negative controlsignals and be clinically used to suppress tumor immune evasion, thusimproving the curative effect of CAR-T in cellular immunotherapy; whenantigen recognition domain is bound to target antigens, signals will betransmitted into cells through chimeric receptors, thereby creating aseries of biological effects such as T cell proliferation, increasedcell factor secretion, increased antiapoptosis secretion, delayed celldeath and target cell lysis.

Preferably, the said chimeric receptor co-stimulator domain employs thecombination of CD28 chimeric receptor co-stimulator as shown in SEQ IDNO: 24 and CD134 chimeric receptor co-stimulator as shown in SEQ ID NO:25.

Preferably, all the said PDL1 single-chain antibody light chain VL, PDL1single-chain antibody heavy chain VH, mesothelin single-chain antibodylight chain VL, mesothelin single-chain antibody heavy chain VH and PDL1single-chain antibody have been humanized.

In the second aspect, the invention provides a preparation method of thesaid OCTS-based CAR-T vector for treating pancreatic cancer andmalignant mesothelioma, including the following steps:

(1) Store on lentiviral skeleton plasmid the AmpR sequence containingampicillin resistance gene as shown in SEQ ID NO: 1, prokaryoticreplicon pUC On sequence as shown in SEQ ID NO: 2, virus replicon SV40Ori sequence as shown in SEQ ID NO: 3, lentiviral packaging cis-elementfor lentiviral packaging, ZsGreen1 green fluorescent protein as shown inSEQ ID NO: 11, IRES ribosome binding sequence as shown in SEQ ID NO: 12,and eWPRE enhanced post-transcriptional regulatory element of Groundhoghepatitis B virus as shown in SEQ ID NO: 13;

(2) Combine into a design scheme for OCTS chimeric receptor the humanEF1α promoter as shown in SEQ ID NO: 14, the said OCTS chimeric receptorstructural domain and PDL1 single-chain antibody as shown in SEQ ID NO:27, and clone the scheme into lentiviral skeleton plasmids by digestion,ligation and recombination to obtain pOCTS-PMs and pOCTS-PMt,recombinant lentiviral plasmids designed by the third-generation OCTS;

(3) Transfect HEK293T/17 cells by obtained recombinant lentiviralplasmids pOCTS-PMs and pOCTS-PMt with lentiviral packaging plasmidspPac-GP and pPac-R and membrane protein pEnv-G respectively. After genetranscription and expression in HEK293T/17 cells, recombinant lentiviralvector packaged successfully will be released into cell culturesupernatants, and then collect supernatants containing recombinantlentiviral vectors;

(4) Purify obtained recombinant lentiviral supernatants with the methodof column purification of filtration, adsorption and elution, and obtainrecombinant lentiviral vectors lvOCTS-PMs and lvOCTS-PMt respectively.

Preferably, in step (4), the said filtration step is to control thevolume of supernatant from 200 ml to 2000 ml, the vacuum degree from−0.5 MPA to 0.9 MPA to prevent the loss of vector caused by blockage.The said adsorption step is to control the PH value of solution from 6to 8 to prevent the vector from inactivating due to the change of pH,and the said elution step is to control the ionic strength of eluentfrom 0.5 M to 1.0M to prevent the change of ionic strength leading toincomplete elution or inactivation of vector.

In the third aspect, the invention provides the application of the saidvectors in the preparation of drugs for treating pancreatic cancer andmalignant mesothelioma.

Compared with existing technologies, the invention has the followingbeneficial effects:

Based on current traditional CAR-T cellular therapy, by optimizing andmodifying the structure of chimeric antigen receptor (CAR), theOCTS-CAR-T technology employed in the invention makes CARs able torecognize two antigens, thereby greatly expanding the recognition scopeof CAR-T cells and making the elimination directed at tumor groups morethorough and with more lasting curative effect, avoids the batch cultureof CAR-T cells, thereby greatly saving costs, avoids multiple reinfusionof different targeting CAR-T cells in patients, thus reducing thespending of patients and odds of recurrence and indirectly improving thelife quality of patients, only needs one transduction with highefficiency in transduction and without prejudice to the curative effectof CAR-T, and does not occupy the precious volume of lentiviraltransgenic vector, which is good for loading other functionalcomponents, and with high efficiency in transgenic vector package andgene transduction.

OCTS, short for One CAR with Two ScFvs, integrates two scFvs into achimeric molecule (as shown in FIG. 1) by means of Series OCTS or TurnOCTS connection, to enable T lymphocyte HLA to independently recognizetwo tumor antigens and recognize more extensive targets compared withtraditional CAR-T cells, thus further extending the scope of eliminationof tumor cells. Basic design of OCTS involves two tumor-associatedantigen (TAA) binding domains (which generally is scFv sector comingfrom monoclonal antibody and antigen binding domain), one extracellularlinker domain, one transmembrane domain, two intracellular signaltransduction domain and one effector domain. ScFv domain is a key factordetermining the specificity and effectiveness of OCTS and the safety ofgene modification of T cells. Upcoming clinical research stage ofOCTS-CAR-T marks that CAR-T in cellular therapy is about to step intoits 2.0 era.

The vector skeleton employed by the invention can be applied to both thesecond-generation and third-generation lentiviral vector structure. Themajor difference in structure between the second-generation andthird-generation lentiviral vectors is shown in FIG. 2B. The inventiongive preference to the third-generation lentiviral vector (as shown inFIG. 2A), removes U3 region from 3′SIN LTR, thereby eliminating thepossibility of self-replication of lentiviral vector and greatlyimproving the security, adds the cPPT and WPRE elements, therebyimproving the transduction efficiency and transgenic expressionefficiency, employs RSV promoter thereby ensuring the continuous andefficient transcription of core RNA in lentiviral vector packaging, andemploys human EF1α promoter, thereby enabling CAR gene to becontinuously expressed in human body for a long time.

In the invention, all the said PDL1 single-chain antibody light chainVL, PDL1 single-chain antibody heavy chain VH, mesothelin single-chainantibody light chain VL, mesothelin single-chain antibody heavy chain VHand PDL1 single-chain antibody have been humanized, and can effectivelyreduce the production of human anti-mouse antibodies (HAMA), extend thehalf life and effect of scFv and increase the lifetime of OCTS-CAR-Tcells.

One or several combinations of co-stimulators used in the invention canenhance the proliferation rate, survival time, killing efficiency,immune memory and other specificities of transduced cells.

OCTS-CAR-T cells employed by the invention can be used for humanclinical trials after being produced by GMP-level workshops.

Recombinant lentiviral vectors employed in the invention can realize theexpression of double target chimeric antigen receptors of thecombination of PDL1, mesothelin, etc. on human T lymphocytes, guide andactivate the killing effect of T lymphocytes on PDL1, mesothelin andother positive cells, and be clinically used to treat pancreatic cancer,malignant mesothelioma and other mesothelin positive/PDL1 positive/bothmesothelin and PDL1 positive malignant tumors.

The invention builds and forms recombinant lentiviral vectors throughrecombinant lentiviral vector skeleton, OCTS structural domain and PDL1single-chain antibody, and recombinant lentiviral vectors obtained insuch way can realize the expression of cellular programmed cell death 1(PDL1) single-chain antibodies in human T lymphocytes, which canefficiently close PDL1, block negative control signals and be clinicallyused to suppress tumor immune evasion, thus improving the curativeeffect of CAR-T in cellular immunotherapy.

It can be seen that OCTS-CAR-T cells said in the invention will providereliable guarantee for tumor cell therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the OCTS chimeric receptor said in theinvention, consisting of the schematic diagram of Series OCTS and TurnOCTS;

FIGS. 2A-2B are schematic diagrams of the structure of the lentiviralvector said in the invention; wherein, FIG. 2A is a schematic diagram ofthe structure of the third-generation lentiviral vector employed by theinvention, and FIG. 2B is a schematic diagram of the structurecomparison of the second and third-generation lentiviral vectors;

FIG. 3 is a flow chart for preparing the recombinant lentiviral vectorin embodiment 1 of the invention; wherein, (A) is a schematic diagram ofthe structure of the lentiviral skeleton plasmid pLenti-3G basic; (B) isa schematic diagram of 2 OCTS plasmids; (C) is a schematic diagram ofthe structure of pPac-GP plasmid; (D) is a schematic diagram of thestructure of pPac-R plasmid; (E) is a schematic diagram of the structureof pEnv-G packaging plasmid;

FIGS. 4A-4C are schematic diagrams of the element order of OCTSstructure in embodiment 1 of the invention; wherein, FIG. 4A is aschematic diagram of the structure of Series OCTS; FIG. 4B is aschematic diagram of the structure of Turn OCTS; FIG. 4C is a schematicdiagram of the list of OCTS Symbols of OCTS structure;

FIGS. 5A-5D show enzyme digestion prediction and enzyme digestionagarose gel electrophoresis diagram of recombinant lentiviral plasmidspOCTS-PMs and pOCTS-PMt in embodiment 1 of the invention; wherein, FIG.5A is a schematic diagram of the enzyme digestion prediction ofpOCTS-PMs, and FIG. 5B is an enzyme digestion agarose gelelectrophoresis diagram of pOCTS-PMs; FIG. 5C is a schematic diagram ofthe enzyme digestion prediction of pOCTS-PMt, and FIG. 5D is an enzymedigestion agarose gel electrophoresis diagram of pOCTS-PMt; in FIG. 5A,lane 1 is 1 kb DNA ladder Marker: the bands from top to bottom are 10kb, 8 kb, 6 kb, 5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb,750 bp, 500 bp, 250 bp; in FIG. 5A, lane 2 is the KpnI enzyme digestionprediction of pOCTS-PMs: the bands from top to bottom are 6254 bp, 4638bp, 1525 bp; in FIG. 5B, lane 1 is the electrophoretic results of 1 kbDNA ladder Marker; in FIG. 5B, lane 2 is the KpnI enzyme digestionelectrophoretic results of pOCTS-PMs; in FIG. 5C, lane 1 is 1 kb DNAladder Marker: the bands from top to bottom are 10 kb, 8 kb, 6 kb, 5 kb,4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp, 250 bp;in FIG. 5C, lane 2 is ApaL I enzyme digestion prediction of pOCTS-PMt:the bands from top to bottom are 5032 bp, 4091 bp, 1731 bp, 1241 bp, 492bp; in FIG. 5D, lane 1 is the electrophoretic results of 1 kb DNA ladderMarker; in FIG. 5D, lane 2 is the enzyme digestion electrophoreticresults of pOCTS-PMt;

FIG. 6 is a schematic diagram of titer detection results of recombinantlentiviral vectors in embodiment 1 of the invention;

FIG. 7 is the flow diagram of the steps of preparing OCTS-CAR-T cellssaid in embodiment 1 of the invention, involving isolated culture,activation, gene transduction, OCTS-CAR-T cell identification and otherstages:

FIG. 8 is a schematic diagram of mycoplasma detection results ofOCTS-CAR-T cells in embodiment 2 of the invention, where lane 1 isDL2000 marker, and the bands from top to bottom are 2 kb, 1 kb, 750 bp,500 bp, 250 bp and 100 bp; lane 2 is a positive control; lane 3 is anegative control; lane 4 is PBS; lane 5 is lysate; lane 6 isOCTS-PMs-CAR-T cell; lane 7 is OCTS-PMt-CAR-T cell;

FIGS. 9A-9D are schematic diagrams of the results of flow cytometry oftransduction efficiency and immunophenotyping of OCTS-CAR-T cell;wherein, FIG. 9A shows the results of transduction efficiency ofOCTS-PMs-CAR-T cell; FIG. 9B shows the results of immunophenotyping ofOCTS-PMs-CAR-T cell, FIG. 9C shows the results of transductionefficiency of OCTS-PMt-CAR-T cell; FIG. 9D shows the results ofimmunophenotyping of OCTS-PMt-CAR-T cell;

FIG. 10 is a schematic diagram of the comparison in terms of killingeffect on different target cells between OCTS-PMs-CAR-T cell andOCTS-PMt-CAR-T cell on different multiplicity of infection conditions inembodiment 3 of the invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The invention is further described below in connection with specificimplementation methods. It should be understood that the specificimplementation methods described herein are expressed by way of examplesand are not constrained by the invention. Without departing from thescope of the invention, the main features of the invention can be usedin various implementation methods.

Materials

1. Lentiviral skeleton plasmid pLenti-3G basic, lentiviral packagingplasmids pPac-GP and pPac-R and membrane protein plasmid pEnv-G,HEK293T/17 cells, homologous recombinase, Oligo Annealing Buffer,MycoAlert Mycoplasma Detection Kit, endotoxin detection kit,PDL1⁺K562,⁺K562, PDL1⁺MESOTHELIN⁺K562 and K562 cells were bought fromShiao (Shanghai) Biomedical Technology Co., Ltd.; specific preparationmethod of lentiviral skeleton plasmid pLenti-3G basic has been disclosedin the patent application No. 201610008360.5 for an invention titled “ACAR-T Transgenic Vector Based on Replication-Competent DefectiveRecombinant Lentiviral vector as Well as Preparation Method andApplication Thereof”;

2. Fresh human peripheral blood provided by healthy donors;

3. Combination of OCTS-PMs and OCTS-PMt DNA sequences designed byShanghai Unicar (see FIG. 4C), synthesized by Shanghai Generay BiotechCo., Ltd. and kept in the form of dry powder of oligonucleotides orplasmid;

4. Tool enzymes Kpn I, ApaL I, Cla I, EcoR I and T4 DNA ligase purchasedfrom NEB;

5. 0.22 μm-0.8 μm PES filter purchased from Millipore;

6. D-PBS (−), 0.4% trypan blue, screen mesh, various cell-culturedishes, culture bags, and culture plates bought from Corning;

7. Opti-MEM, Pen-Srep, Hepes, FBS, AIM-V, RPMI 1640, DMEM, lipofectamine3000 bought from Invitrogen;

8. Biotinylated protein L bought from GeneScript;

9. LDH detection kits bought from Promega;

10. Ficoll lymphocyte separation medium bought from GE;

11. 20% human albumin solution bought from CSL Behring;

12. CryoPremium freezing medium and sorting buffer solution bought fromShanghai Unicar;

13. rIL-2, rIL-7, rIL-15, rIL-21 purchased from peprotech;

14. CD3 monoclonal antibody, CD28 monoclonal antibody, CD3/CD28 magneticbead, CD4/CD8 magnetic bead bought from a German company Miltenyi;

15. Refrigerated centrifuge bought from an American companyThermoScientific;

16. FACS bought from Thermo;

17. Fluorescence inversion microscope system bought from Olympus;

18. CD4-FITC and CD8-APC bought from BioLegend;

19. 0.9% saline solution bought from Jinmai;

20. ProteinL Magnetic Beads bought from BioVision;

21. PrimeSTAR and RetroNectin bought from Takara;

22. phycoerythrin (PE)-conjugated streptavidin bought from BDBioscience;

23. Plasmid extraction kits and agarose gel recovery kits purchased fromMN:

24. TOP 10 competent cells purchased from Tiangen;

25. NaCl, KCl, Na₂HPO₄.12H₂O, KH₂PO₄, Trypsin, EDTA, CaCl₂, NaOH,PEG6000 purchased from Shanghai Sangon Biotech;

26. DNeasy kits purchased from Shanghai Generay;

27. SA-HRP purchased from Shanghai Yeasen;

28. Primers: Primers required for amplifying DNA fragments and targetsites, designed by primer design principle and synthesized by ashanghai-based biotechnology company specifically as follows:

EF1α-F: (SEQ ID NO. 28) 5′-ATTCAAAATTTTATCGATGCTCCGGTGCCCGTCAGT-3′EF1α-R: (SEQ ID NO. 29) 5′-TCACGACACCTGAAATGGAAGA-3′ OCTS-F:(SEQ ID NO. 30) CATTTCAGGTGTCGTGAGGATCCGCCACCATGGCGCTGCCGGTGAC OCTS-R:(SEQ ID NO. 31) GGGGAGGGAGAGGGGCTTAGCGCGGCGGCAGCG IRES-F:(SEQ ID NO. 32) GCCCCTCTCCCTCCCCC IRES-R: (SEQ ID NO. 33)ATTATCATCGTGTTTTTCAAAGGAA PDL1scab-F: (SEQ ID NO. 34)AAAACACGATGATAATGCCACCATGAACTCCTTCTCCACAAGCG PDL1scab-R: (SEQ ID NO. 35)AATCCAGAGGTTGATTGTCGACGAATTCTCATTTGCCCGGGCTCAG WPRE-QPCR-F:(SEQ ID NO. 36) 5′-CCTTTCCGGGACTTTCGCTTT-3′ WPRE-QPCR-R: (SEQ ID NO. 37)5′-GCAGAATCCAGGTGGCAACA-3′ Actin-QPCR-F: (SEQ ID NO. 38)5′-CATGTACGTTGCTATCCAGGC-3′ Actin-QPCR-R: (SEQ ID NO. 39)5′-CTCCTTAATGTCACGCACGAT-3′

29. In the invention, the said DNA fragments shown in SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26,SEQ ID NO: 27 were synthesized by Shanghai Generay Biotech Co., Ltd. inline with sequences provided by the inventor thereof.

Embodiment 1. Preparation of OCTS-CAR-T Cells

I. Methods of Preparation, Purification and Detection of RecombinantLentiviral Vectors lvOCTS-PMs and lvOCTS-PMt

The preparation method of recombinant lentiviral vectors described inthe invention is as follows (see FIG. 3):

1. Combine into a design scheme for OCTS chimeric receptor the humanEF1α promoter (SEQ ID NO: 14), OCTS structures [OCTS-PMs and OCTS-PMt],CD8 leader chimeric receptor signal peptide (SEQ ID NO: 15), PDL1single-chain antibody light chain VL (SEQ ID NO: 16), PDL1 single-chainantibody heavy chain VH (SEQ ID NO: 17), mesothelin single-chainantibody light chain VL (SEQ ID NO: 18), mesothelin single-chainantibody heavy chain VH (SEQ ID NO: 19), antibody Inner-Linker (SEQ IDNO: 20), single-chain antibody Inter-Linker (SEQ ID NO: 21), CD8 Hingechimeric receptor linker (SEQ ID NO: 22), CD8 Transmembrane chimericreceptor transmembrane domain (SEQ ID NO: 23), CD28 chimeric receptorco-stimulator (SEQ ID NO: 24), CD134 chimeric receptor co-stimulator(SEQ ID NO: 25), TCR chimeric receptor T cell activation domain (SEQ IDNO: 26), PDL1single-chain antibody (SEQ ID NO: 27), and clone the schemeinto the lentiviral skeleton plasmid pLenti-3G basic by digestion,ligation and recombination to obtain recombinant lentiviral plasmidspOCTS-PMs and pOCTS-PMt respectively (see FIGS. 4A-4C for element orderand No.).

(1) Conduct double digestion of the lentiviral skeleton plasmidpLenti-3G basic with Cla I and EcoR I restriction enzymes,electrophorese the product thereof on a 1.5% agarose gel to confirm the5823 bp fragment VI, and then recover and place such gel in an Eppendorftube, and recover corresponding fragments with the agarose gel recoverykit of MN (see Table 1) and determine the purity and concentration ofthe product thereof;

TABLE 1 Procedures for the recovery of agarose gel 1. Sol Add the solsolution in a ratio of 200 μl NTI/100 mg gel, and place it in a 50° C.water bath for 5-10 minutes. 7. Bind to Centrifuge at 11,000 g for 30seconds, and DNA discard the filtrate. 8. Wash Add 700 μl NT3,centrifuge at 11,000 g for membrane 30 seconds, and discard the filtrate9. Wash Repeat step 3 once membrane 10. Dry Centrifuge at 11,000 g for 1minute, replace with a new collection tube, and leave it at roomtemperature for 1 minute. 11. Elute Add 15-30 μl NE, leave it at roomtemperature DNA for 1 minute, centrifuge at 11,000 g for 1 minute, andthen collect the filtrate.

(2) Use the primers EF1α-F and EF1α-R with the synthesized human EF1αpromoter (SEQ ID NO: 14) as a template, apply the system in Table 2 onthe PCR circulation conditions: 98° C. 3 min, (98° C. 10 sec, 55° C. 15sec, 72° C. 2 min)*35 cycle, 72° C. 10 min, electrophorese the productthereof on a 1.5% agarose gel to confirm the 1208 bp fragment a, andthen recover and place such gel in an Eppendorf tube, and recovercorresponding fragments with the agarose gel recovery kit of MN (seeTable 1) and determine the purity and concentration of the productthereof;

TABLE 2 50 μl PCR reaction system Reagent Volume (μl) H₂O 32.5 5× Buffer(with Mg2+) 10 dNTP (2.5 mM each) 4 Primer1 (+)(10 μM) 1 Primer2 (−)(10μM) 1 Template 1 PrimeSTAR 0.5

(3) Use the primers OCTS-F and OCTS-R with the synthesized OCTS-PMs as atemplate, apply the system in Table 2 on the PCR circulation conditions:98° C. 3 min, (98° C. 10 sec, 55° C. 15 sec, 72° C. 30 sec)*3522 cycle,72° C. 5 min, electrophorese the product thereof on a 1.5% agarose gelto confirm the 2370 bp fragment b, and then recover and place such gelin an Eppendorf tube, and recover corresponding fragments with theagarose gel recovery kit of MN (see Table 1) and determine the purityand concentration of the product thereof;

(4) Use the primers OCTS-F and OCTS-R with the synthesized OCTS-PMt as atemplate, apply the system in Table 2 on the PCR circulation conditions:98° C. 3 min, (98° C. 10 sec, 55° C. 15 sec, 72° C. 30 sec)*35 cycle,72° C. 5 min, electrophorese the product thereof on a 1.5% agarose gelto confirm the 2382 bp fragment c, and then recover and place such gelin an Eppendorf tube, and recover corresponding fragments with theagarose gel recovery kit of MN (see Table 1) and determine the purityand concentration of the product thereof;

(5) Use the primers IRES-F and IRES-R with the synthesized IRES ribosomebinding sequence (SEQ ID NO: 12) as a template, apply the system inTable 2 on the PCR circulation conditions: 98° C. 3 min, (98° C. 10 sec,55° C. 15 sec, 72° C. 30 sec)*35 cycle, 72° C. 5 min, electrophorese theproduct thereof on a 1.5% agarose gel to confirm the 575 bp fragment d,and then recover and place such gel in an Eppendorf tube, and recovercorresponding fragments with the agarose gel recovery kit of MN (seeTable 1) and determine the purity and concentration of the productthereof;

(6) Use the primers PDL1scab-F and PDL1 scab-R with the synthesized PDL1single-chain antibody (SEQ ID NO: 27) as a template, apply the system inTable 2 on the PCR circulation conditions: 98° C. 3 min, (98° C. 10 sec,55° C. 15 sec, 72° C. 30 sec)*35 cycle, 72° C. 5 min, electrophorese theproduct thereof on a 1.5% agarose gel to confirm the 2355 bp fragment c,and then recover and place such gel in an Eppendorf tube, and recovercorresponding fragments with the agarose gel recovery kit of MN (seeTable 1) and determine the purity and concentration of the productthereof;

(7) Add combinations of recombinant lentiviral plasmid DNA fragmentsinto the Eppendorf tubes with a total volume of 5 μL and at a molarratio of 1:1:1; add 15 μL homologous recombinant enzyme reactionsolution into the tubes, mix and incubate them at 42° C. for 30 minutes,and put the tubes on ice for 2-3 minutes; add the reaction solution into50 μL TOP10, rotate the tubes gently to mix the contents, place thetubes in ice for 30 minutes, put the tubes in a constant temperaturewater bath pot preheated to 42° C. for 90-second heat shock, quicklytransfer the tubes to the ice bath, and cool the cells for 2-3 minutes;add 900 μl LB culture medium into each tube, transfer the tubes to ashaking bed at 37° C., and incubate the tubes for 1 hour to resuscitatethe bacteria; take 100 μL transformed bacteria solution, coat it on theAmp LB agar plate, invert the flat dish, and put it in a constanttemperature incubator at 37° C. for 16-hour cultivation.

TABLE 3 Combination of recombinant lentiviral plasmid DNA fragmentsRecombinant Combination lentiviral plasmid of fragments pOCTS-PMs a, b,d, e pOCTS-PMt a, c, d, e

Select clones for colony PCR identification, among which thoseidentified as correct are exactly recombinant lentiviral plasmidspOCTS-PMs and pOCTS-PMt, conduct enzyme digestion identification ofcorrect clones (see FIGS. 5A-5D), and send the sequencing reviewresults.

2. Packaging of Recombinant Lentiviral Vectors lvOCTS-PMs and lvOCTS-PMt

(1) Complete medium: take out the pre-heated fresh medium, add 10% FBS+5ml Pen-Srep, and mix them upside down;

(2) 1×PBS solution: weigh 8 g of NaCl, 0.2 g of KCl, 3.58 g ofNa₂HPO₄.12H₂O, 0.24 g of KH₂PO₄, and put them in a 1000 ml beaker, andadd 900 ml of Milli-Q grade ultrapure water to dissolve. Aftercompletion of the dissolution, the volume was adjusted to 1000 ml usinga 1000 ml measuring cylinder, and the mixture was sterilized by heatsterilization at 121° C. for 20 minutes;

(3) 0.25% Trypsin solution: weigh 2.5 g of Trypsin, 0.19729 g EDTA, andput them in a 1000 ml beaker, and add 900 ml of 1×PBS solution todissolve. After completion of the dissolution, the volume was adjustedto 1000 ml using a 1000 ml measuring cylinder, and the mixture wassterilized via 0.22 μM filter. It could be saved in the refrigerator at−20° C. for long-term use;

(4) 0.5 M CaCl₂) solution: weigh 36.75 g of CaCl₂, and dissolve it with400 ml of Milli-Q grade ultrapure water; The volume was adjusted to 500ml with Milli-Q grade ultrapure water, and mixed; the mixture wassterilized via 0.22 μM filter, and stored in 50 ml centrifuge tubes withabout 45 ml in each tube at 4° C.;

(5) 2×HBS solution: weigh 4.09 g of NaCl, 0.269 g of Na₂HPO₄, 5.96 g ofHepes, and dissolve them with 400 ml Milli-Q grade ultrapure water;After calibrating the pH meter, the pH of the HBS solution was adjustedto 7.05 with 2 M NaOH solution. It was about 3 ml of 2 M NaOH to consumeto adjust the pH of each bottle of HBS;

(6) The frozen HEK293T/17 cells were removed from the liquid nitrogencontainer and rapidly transferred to a 37° C. water bath for 1-2minutes, and then put them on a super clean bench. Aseptically transferall the liquid in the freezing tube to a 10 cm² petri dish, and make upDMEM containing 10% FBS to 8 mL/10 cm² dish, and observe the cells undermicroscope after 24 hours. Passage was performed with the degree of cellconfluence greater than 80%;

(7) HEK293T/17 cells with good cell status and no pollution wereselected, and each 2-6 petri dishes were used as a group. Aftertrypsinizing the cells, 4-12 ml of complete medium was pipetted with anelectric pipette to add 2 ml to each digested dish to avoid drying thedish; all cells were isolated into single cell suspensions using a 1 mlpipette and transferred to medium bottles;

(8) The remaining cells in the above 2-6 petri dishes were transferredto the medium bottles, and the petri dishes were rinsed with the mediumagain;

(9) Close the cap of the medium bottles and turn them upside down forabout 10 times to fully mixed the cell suspension. Transfer the cells to8-24 10 cm² petri dishes. For each dish, there shall be about 4×10⁶cells/10 ml complete medium. If the cell density is significantlydifferent from the expected, the number of cells is required to becounted, and then the cells will be inoculated according to the quantityof 4×10⁶ per dish;

(10) Arrange each of the 6 petri dishes into a pile, and keep the fitbetween the upper and lower dishes. Shake the petri dishes left andright, back and forth several times to make cells fully spread out, andthen put them into an incubator with 5% CO₂. The remaining cells weretreated as the same;

(11) Upon Checking the passage cells, the cells shall be at 70-80%confluence, with full contour, good attachment and even distribution inpetri dishes;

(12) For changing the solution, the medium was replaced with freshcomplete medium with 9 ml per dish. The CO₂ concentration of incubatorwas increased to 8%;

(13) To prepare DNA/CaCl₂ according to N+0.5. The amount of HEK293T/17cell transfection plasmid per dish was used in the following ratios:recombinant lentiviral plasmid (20 μg), pPac-GP (15 μg), pPac-R (10 μg),pEnv-G (7.5 μg). Take a new 5 ml centrifuge tube, add 0.5 M CaCl₂: 0.25ml, recombinant lentiviral plasmid 20 μg: pPac-GP 15 μg: pPac-R 10 μg:pEnv-G 7.5 μg, supplement ultrapure water to 0.5 ml, and cover the capto mix them fully;

(14) Take another 5 ml centrifuge tube and add 0.5 ml DNA/CaCl₂)solution. Open a vortex mixer, hold the upper end of the 5 ml centrifugetube with one hand, and make the bottom of the tube contact theoscillation chamber, so that the liquid could spread on the tube wall.Take a 1 ml pipette with anther hand to suck 0.5 mL 2×HBS solution, addit into the centrifuge tube slowly and control the flow velocity. It wasadvisable to complete the drip in half a minute. After 2×HBS was added,it should be oscillated for another 5 seconds, and then stoposcillating. It could be directly added into the cells that needtransfection;

(15) Take a dish of cells and drop 1 mL calcium transfection solution inthe centrifuge tube in the dish to distribute the calcium transfectionsolution throughout the petri dish as much as possible;

(16) After the calcium transfection solution was added, the petri dishwas marked on the cover, and put back in another incubator with 5% CO₂.Make sure that the petri dish was placed horizontally, and that therewere no more than 6 petri dishes in each pile. These dishes were placedin the incubator with 5% CO₂ for 6-8h;

(17) The CO₂ concentration of the first incubator was adjusted at 5%;

(18) The cells status was check 24 hours later. The cell confluenceshould be around 80-85% and in good condition. Aspirate the medium andreplace 10 ml of fresh DMEM complete medium;

(19) The transfection efficiency was observed 48 hours later. Most cellswere still adherent. It could be seen that more than 95% of the cellswould have green fluorescence. The supernatant of the same viruspackaging was collected together, and 10 mL of fresh medium was added tothe petri dish;

(20) The same virus supernatant was collected again 72 hours later. Thetwo collections were put together, and the petri dishes were discarded;the supernatant collected at this time contained the recombinantlentiviral vectors lvOCTS-PMs and lvOCTS-PMt.

3. Purification of Recombinant Lentiviral Vectors by Ion ExchangeChromatography

(1) The collected supernatant was filtered through a 0.22 μm-0.8 μm PESfilter using a Thermo vacuum pump to remove impurities;

(2) 1.5 M NaCl 250 mM Tris-HCl (PH6-8) was added to the supernatant at aratio of 1:1 to 1:10;

(3) Two ion exchange columns were placed in series, and they were passedthrough sequentially by 4 ml 1 M NaOH, 4 ml 1 M NaCl, 5 ml 0.15 M NaCl25 mM Tris-HCl (pH 6-8) solution;

(4) The solution obtained in step 2 was pumped into the ion exchangecolumn with a peristaltic pump at a rate of 1-10 m/min

(5) After all the supernatant was passed through the column, it waswashed with 10 ml of 0.15 M NaCl 25 mM Tris-HCl (pH 6-8) solution;

(6) According to the sample size, 1-5 ml of 1.5 M NaCl 25 mM Tris-HCl(pH 6-8) was used for elution and the eluate was collected; and

(7) The eluate was divided into tubes about 25 to 50 μl each, and storedin a refrigerator with −80° C. for long-term storage.

4. Titre Determination of Recombinant Lentiviral Vectors

(1) 293T cells were inoculated with 24-well plates. The number of cellsin each well was 5×10⁴, and the volume of medium added was 500 μL. Asthe growth rate of different types of cells was different, the rate ofcell fusion during viral infection was 40%-60%;

(2) Three sterile EP tubes were prepared, and 90 μL fresh completemedium (high glucose DMEM+10% FBS) was added into each tube to inoculatethe cells. 24 hours later, the cells in the two pores were taken andcounted with a hemocytometer to determine the actual number of cells atthe time of infection, denoted as N;

(3) 10 μL of the virus stock to be determined was added to the firsttube. After gently mixing, 10 μL of the virus stock was added to thesecond tube, and then sequentially operated until the last tube; 410 μLcomplete medium (high glucose DMEM+10% FBS) was added into each tube,and the final volume was 500 μL;

(4) 20 hours after the infection, the cultural supernatant was removedand replaced with 500 μL complete medium (high glucose DMEM+10% FBS).The cells were continuously cultured for 48 hours in 5% CO₂;

(5) After 72 hours, the fluorescence expression was observed. Undernormal circumstances, the number of fluorescence cells decreased withthe increase of dilution ratio. At the same time, photos were taken;

(6) The cells were digested with 0.2 ml 0.25% trypsin-EDTA solution, andthen they were placed at 37° C. for 1 minute. The whole cellular surfacewere purged with medium, and the cells were collected by centrifugation.Genomic DNA was extracted according to the instructions of DNeasy kit.200 μL of eluent were added to each sample tube to remove DNA, and thenthey were quantified,

(7) The DNA detection qPCRmix manifold I was prepared (QPCR primersequences were SEQ ID NO: 36-SEQ ID NO: 37):

2× TaqMan Master Mix  25 μl × n Forward primer (100 pmol ml-1) 0.1 μl ×n Reverse primer (100 pmol ml-1) 0.1 μl × n Probe (100 pmol ml-1) 0.1 μl× n H₂O 19.7 μl × n 

n=number of reactions. For example, the total n were 40. 1 ml of 2×TaqMan Universal PCR Master Mix, 4 μl of forward primer, 4 μl of reverseprimer, 4 μL of probe and 788 μL of H₂O were mixed and placed on iceafter being shaken;

(8) The reference DNA detection qPCRmix manifold II were prepared (QPCRprimer sequences were SEQ ID NO: 38-SEQ ID NO: 39):

2× TaqMan Master Mix 25 μl × n 10× RNaseP primer/probe mix 2.5 μl × nH₂O 17.5 μl × n 

n=number of reactions. For example, the total n were 40. 1 ml of 2×TaqMan Universal PCR Master Mix, 100 μL pf 10×RNaseP primer/probe mixand 700 μL of H₂O were mixed and placed on ice after being shaken;

(9) The PCR system was established on a pre-cooled 96-well PCR plate.Take 45 μL from each tube of manifold I to add to the wells of each rowof A-D. Take 45 μL from each tube of manifold II to add to the wells ofeach row of E-G.

(10) 5 μL of the standard plasmid and the genomic DNA from the samplesto be tested were taken respectively to add to the A-D row, and eachsample was repeated once. 1 well was left to add 5 μL of water asno-template control.

(11) 5 μl of the genomic standards and the genomic DNA from the samplesto be tested were taken respectively to add to the E-G row, and eachsample was repeated once. 1 well was left to add 5 μL of water asno-template control.

(12) The quantitative PCR instrument used was the ABI PRISM 7500quantitative system. The cyclic conditions were set to: 50° C. 2 min,95° C. 10 min, (95° C. 15 sec, 60° C. 1 min)×40 cycle.

Data analysis: the copy number of lentiviral vectors integrated in themeasured DNA samples was calibrated with the number of genomes to obtainthe copy number of viruses integrated in each genome.

The calculation formula of integration units per ml (IU ml⁻¹) was asfollows:IU ml⁻¹=(C×N×D×1000)/Vwherein: C=the average virus copy number per genome integration

-   -   N=number of cells at the time of infection (approximately 1×10⁵)    -   D=dilution of the viral vector    -   V=the volume of diluted virus added

(13) Titer results of recombinant lentiviral vectors lvOCTS-PMs andlvOCTS-PMt (see FIG. 6).

II. Preparation of OCTS-CAR-T Cell

Preparation method of the OCTS-CAR-T cell described in the invention isas below (see FIG. 7):

1. PBMC Separation

(1) Draw 50 ml fresh peripheral blood from healthy donors;

(2) Spray ethyl alcohol onto the blood collecting bag twice and wipe itoff;

(3) Draw blood cells out of the bag with a 50 ml injector and transferthem to a new 50 ml tube;

(4) Centrifuge the tube at 400 g, 20° C. for 10 min;

(5) Transfer upper blood plasma into a new 50 ml centrifuge tube,inactivate the blood plasma at 56° C. for 30 min, centrifuge the tube atindoor temperature, 2000 g for 30 min, and take and put the supernatantinto a 50 ml centrifuge tube for later use;

(6) Add D-PBS(−) into the tube until the volume of the solution in itreaches 50 ml, tighten up the cap, and overturn the tube for mixing thesolution in it;

(7) Take 2 new 50 ml centrifuge tubes, and add 15 ml Ficoll into each ofthem;

(8) Carefully add 25 ml blood cell diluent into the Ficoll in each tube,and centrifuge the tubes at 800 g, 20° C. for 20 min;

(9) There are four layers of liquid in the centrifuge tubes, which fromtop to bottom are yellow blood plasma layer (recovered for later use),albuginea, water white Ficoll, red and black mixed cells;

(10) Carefully draw the albuginea into a new 50 ml centrifuge tube, addD-PBS(−) into the tube until the volume of the solution in it reaches 50ml, overturn the tube for mixing the solution in it, and centrifuge thetube at 500 g, 20° C. for 10 min;

(11) Add 25 ml 5% human serum albumin, resuspend cells, and centrifugethe tube at 400 g, 20° C. for 10 min;

(12) Remove the supernatant, add 25 ml 5% human serum albumin, resuspendcells until they are precipitated, and count after getting them throughthe 70 um screen mesh; and

(13) Take a portion of cell suspension containing 1.25×10⁸ cells foractivation, centrifuge the remaining cell suspension at 400 g, 20° C.for 10 min, add CryoPremium into and freeze it.

2. CD4/CD8 Positive T Cell Sorting

(1) Count obtained PBMCs, add sorting buffer solution in the ratio of 80μL/10⁷ cells, and resuspend cells until they are precipitated;

(2) Add CD4/CD8 magnetic beads in the ratio of 20 μL/10⁷ cells, andincubate the solution at 4° C. for 15 min after blowing and mixing it;

(3) Take magnetic beads out of the cell mixture, add sorting buffersolution into it in the ratio of 2 ml/10⁷ cells, overturn and mix it,and centrifuge it at 250 g, 4° C. for 10 min; (4) Add sorting buffersolution in the ratio of 500 μL/10s cells, and resuspend cells untilthey are precipitated;

(5) Clamp the LS separation column onto the magnetic shelf withtweezers;

(6) Prepare 2 15 ml centrifuge tubes at the same time, and mark themCD4−/CD8−cell sap (tube A) and CD4+/CD8+cell sap (tube B) respectively;

(7) Rinse LS with 3 ml separation buffer solution, and use tube A tocollect the buffer solution;

(8) Add the cell-magnetic bead mixture, and then 3 ml buffer solution towash the column three times (new liquid is added when there is no liquidresidue), and collect obtained CD4/CD8-cells;

(9) Separate the LS separation column from the magnetic shelf, use tubeB to collect cell suspension, add 5 ml buffer solution, wash the columntogether with the inner plunger thereof a little harder, collectCD4+/CD8+cells, take samples from them and count the samples; and

(10) Resuspend cells until they are precipitated with AIM-V mediumaccording to the cell density of 1×10⁶/ml-4×10⁴/ml, and add 2×10⁵-1-10⁶U/L IFN-γ factors.

3. Activation of T Cells

(1) Add into a 24-pore plate one day in advance the 1-10³ ug/L-1×10⁴ug/L CD3 monoclonal antibody and 1×10³ ug/L-1×10⁴ ug/L CD28 monoclonalantibody, seal it with sealing film, and coat it overnight at 4° C.; and

(2) Take out the coated T75 bottle, discard the coating buffer, and washit with D-PBS(−) once; inject the cell suspension obtained by sortinginto a T75 bottle, share it up, and put it in an incubator at 37° C., 5%CO₂ for culture;

4. CAR Gene Transduction and OCTS-CAR-T Cell Induction Culture

(1) Cost 1×10³ ug/L-1×10⁴ ug/L RetroNectin in a 24-pore plate one day inadvance, seal it with sealing film, and coat it overnight at 4° C.; and

(2) Add lentiviral transgenic vectors lvOCTS-PMs and lvOCTS-PMt into the24-pore plate according to 5×10⁵ cells for each pore and MOI=5-20 alongwith AIM-V medium containing 2×10⁵-5×10⁵ U/L rIL-2, 5×10³ ng/L-1×10⁴ng/L rIL-7, 5×10³ ng/L-1×10⁴ ng/L rIL-15, 5×10³ ng/L-1×10⁴ ng/L rIL-21and 10% autoserum for continuous culture at 37° C., 5% CO₂.

5. In-Vitro Expansion of OCTS-CAR-T Cells

(1) Add the same amount of AIM-V medium containing 2×10⁵-5×10⁵ U/LrIL-2, 5×10³ ng/L-1×10⁴ ng/L rIL-7, 5×10³ ng/L-1×10⁴ ng/L rIL-15, 5×10³ng/L-1×10⁴ ng/L rIL-21 and 10% autoserum every 2 days, keep PH within6.5-7.5 and cell density within 5×10⁵-2-10⁶/ml, and continue to cultureit at 37° C., 5% CO₂ for 10-14 days; and

(2) Use the frozen and cultured OCTS-CAR-T cells for subsequentdetection on the 7^(th) day or so.

Embodiment 2. OCTS-CAR-T Cell Pathogen Detection and ExpressionDetection

I. Endotoxin Determination

(1) The working standard of endotoxin was 15 EU per dose;

(2) Sensitivity of Tachypiens Amebocyte Lysate (TAL) λ=0.25 EU/ml, 0.5ml/tube

(3) Dilution of endotoxin standard: take one endotoxin standard, diluteit into 4λ and 2λ solution with BET water, seal with sealing film andvortex for 15 min; During dilution, each dilution step should be mixedon the vertex mixer for 30 s;

(4) Adding samples: Several TAL were taken, each was dissolved in 0.5 mlof BET water, and then divided into several endotoxin-free tubes (0.1 mleach tube). Two of them were negative control which were added 0.1 ml ofBET water to each of them;

Two tubes were used for positive control which were added 0.1 mlendotoxin working standard solution with concentration of 2λ to each ofthem;

Two tubes were used for positive control of sample which were added 0.1ml sample solution contained 2λ endotoxin standard (1 ml of 20× dilutionof sample to be tested+1 ml of solution contained 4λ endotoxinstandard=2 ml of 40× dilution of sample contained 2λ endotoxinstandard).

0.1 ml sample was added into the sample tube at the dilution rate shownin Table 4 and with 37±1° C. water bath (or incubator) and heatpreservation for 60±1 min;

TABLE 4 Dilution ratio of endotoxin and corresponding endotoxin contentDilution Original Multiple Fluid 5 10 20 40 80 160 Corresponding 0.251.25 2.5 5 10 20 40 EU/ml Results

(5) According to the endotoxin detection results of OCTS-CAR-T cells (asshown in Table 5), the content of endotoxin in all cells was less than2.5 EU/ml, meeting the standard in the Pharmacopoeia of the People'sRepublic of China, which is less than 10 EU/ml;

TABLE 5 Original Dilution Multiple Fluid 5 10 20 40 80 160 Corresponding0.25 1.25 2.5 5 10 20 40 EU/ml OCTS-PMs-CAR-T (+) (−) (−) (−) (−) (−)(−) OCTS-PMt-CAR-T (+) (+) (−) (−) (−) (−) (−)

II. Detection of Mycoplasma

(1) Three days before the experiment, the cell samples were cultured inantibiotic-free medium:

(2) 1 ml cell suspension (more than 1*10⁵ cells) was collected andplaced in a 1.5 ml centrifugal tube;

(3) Centrifuge at 13,000 g for 1 min, collect sediment and discardculture medium;

(4) Add 500 μL PBS, blowing or whirlpool oscillation with the gun head,and resuspend sediment. Centrifuge for 5 min at 13,000 g;

(5) Repeat step (4) once;

(6) Add 50 μL Cell Lysis Buffer, blow and suck with gunhead, mix well,and incubate in water bath at 55° C. for 20 minutes;

(7) The samples were heated at 95° C. for 5 minutes;

(8) After centrifugation at 13,000 g for 5 min, 5 μL supernatant wasused as template. The 25 μL PCR reaction system was ddH20 6.5 μL, MycoMix 1 μL, 2× Taq Plus Mix Master (Dye Plus) 12.5 μL and template 55 μL.The cycle conditions of PCR were 95° C. 30 sec, (95° C. 30 sec, 56° C.30 sec, 72° C. 30 sec)*30 cycle and 72° C. 5 min; and

(9) According to the mycoplasma detection results (as shown in FIG. 8),the OCTS-CAR-T cells contained no mycoplasma.

III. OCTS Gene Transduction Efficiency Detection and ImmunophenotypingDetection

(1) Collect T cells transduced by virus, resuspend cells with D-PBS(−)solution containing 1-4% human serum albumin, and adjust the ratio to1×10⁶/ml;

(2) Add D-PBS(−) solution containing 1-4% human serum albumin into acentrifuge tube, mix it, centrifuge it at 350 g for 5 min, and discardthe supernatant;

(3) Repeat step (2) once;

(4) Resuspend cells with 0.2 ml D-PBS(−) solution containing 1-4% humanserum albumin, add into the centrifuge tube 1 μL 1 mg/μL protein L, 5 μLCD4−FITC and 5 μL CD8−APC, mix it, and incubate it at 4° C. for 45 min;

(5) Add into the centrifuge tube 1 ml D-PBS(−) solution containing 1-4%human serum albumin, mix it, centrifuge at 350 g for 5 min and discardthe supernatant;

(6) Repeat step (5) twice;

(7) Resuspend cells with 0.2 ml D-PBS(−) solution containing 1-4% humanserum albumin, add 0.2 μL PE-SA into the centrifuge tube, mix it, andincubate it at 37° C. in dark for 15 min;

(8) Add into the centrifuge tube 1 ml D-PBS(−) solution containing 1-4%human serum albumin, mix it, centrifuge at 350 g for 5 min and discardthe supernatant;

(9) Resuspend cells with 1 ml D-PBS(−) solution until they areprecipitated, centrifuge at 350 g for 5 min and discard the supernatant;

(10) Repeat step (9) twice;

(11) Resuspend cells with 0.4 ml D-PBS(−) solution until they areprecipitated, and detect the cells with FACS; and

(12) According to the results of OCTS gene transduction efficiencydetection and immunophenotyping detection shown in FIGS. 9A-9D, theefficiency of infection of most prepared OCTS-CAR-T cells were within30%-40%, and the ratio of CD4 positive cells to CD8 positive cells waswithin 1:3-3:1, meaning subsequent function detection can be carriedout.

Embodiment 3. Functional Detection of OCTS-CAR-T Cell

I. Evaluation of Killing Effect on Target Cells

(1) Respectively culture target cells [PDL1⁺K562, ⁺K562,PDL1⁺MESOTHELIN⁺K562, K562 cells] and effector cells [OCTS-CAR-T cells];

(2) Collect target cells 4×10⁵ cells and OCTS-CAR-T cells 2.8×10⁶ cells,centrifuge them at 800 g for 6 min, and discard the supernatant;

(3) Respectively resuspend target cells and effector cells with 1 mlD-PBS(−), centrifuge them at 800 g for 6 min, and discard thesupernatant;

(4) Repeat step (3) once;

(5) Resuspend effector cells with 700 μL medium (AIM-V medium+1-10%FBS), and target cells with 2 ml medium (AIM-V medium+1-10% FBS);

(6) Set up the experimental pores with the multiplicity of infection of1:1, 5:1 and 10:1, and control groups (K562 cells) with 3 compound poreseach. Grouping of co-incubation of effector cells respectively withsingle target cells and double target cells is shown in Table 6;

TABLE 6 Effector Target Cell Cell 1 Target Cell 2 Target Cell 3OCTS-PMs- PDL1⁺ MESOTHELIN⁺ PDL1⁺ CAR-T K562 K562 MESOTHELIN⁺ K562OCTS-PMt- PDL1⁺ MESOTHELIN⁺ PDL1⁺ CAR-T K562 K562 MESOTHELIN⁺ K562

(7) Plate centrifugation at 250 g for 5 min;

(8) Culture it in an incubator at 37° C., 5% CO₂ for 4 hours,

(9) Plate centrifugation at 250 g for 5 min;

(10) Take 50 μL supernatant from each pore to a new 96-pore plate, andadd 50 μL substrate solution to each pore (in dark);

(11) Incubate it in dark for 25 min;

(12) Add 50 μL stop buffer into each pore;

(13) Detect 490 nm absorbance with microplate reader;

(14) Average the three compound pores; reduce the light absorption valueof all experimental pores, target cell pores and effector cell pores bythe average light absorption value under the background of medium;reduce the maximum light absorption value of target cells by the averagevolume correction control light absorption value;

(15) Bring the corrected values obtained in step (14) into the followingformula to calculate the percentage of cytotoxicity produced by eachmultiplicity of infection. According to the results shown in FIG. 10,OCTS-CAR-T cells have better killing effect on their own single targetcells and double target cells, and the killing efficiency of CAR-T cellswith Turn OCTS structure is lightly higher than that of CAR-T cells withSeries OCTS structure;Killing efficiency=(Experimental pore−Effector cell pore−Target cellpore)/(Maximum target cell pore−Target cell pore)×100%

(16) Such experimental results show that OCTS structure formed bymodifying the antigen recognition domain in traditional CAR structurecan significantly improve the scope of recognition and killing targetcells of OCTS-CAR-T cells, and thus OCTS-CAR-T cells will play a hugerole in future cellular therapy of pancreatic cancer, malignantmesothelioma and other mesothelin positive/PDL1positive/both mesothelinand PDL1 positive malignant tumors.

What is claimed is:
 1. An OCTS-based CAR-T vector for targetingmalignant tumors expressing mesothelin, PDL1, or both of the mesothelinand the PDL1, comprising: a lentiviral skeleton plasmid, a human EF1αpromoter, a first nucleotide sequence of a one CAR with two ScFvs (OCTS)chimeric receptor structural domain, and a second nucleotide sequenceencoding a programmed cell death receptor ligand 1 (PDL1) single-chainantibody; wherein the lentiviral skeleton plasmid comprises: an AmpRsequence containing an ampicillin resistance gene for a vast expansionof a target bacterial strain, wherein the AmpR sequence is shown in SEQID NO: 1; a prokaryotic replicon pUC Ori sequence for plasmidreplication, wherein the prokaryotic replicon pUC Ori sequence is shownin SEQ ID NO: 2; a viral replicator SV40 Ori sequence for enhancingreplication in eukaryotic cells, wherein the viral replicator SV40 Orisequence is shown in SEQ ID NO: 3; a lentiviral packaging cis-elementfor lentiviral packaging; a third nucleotide sequence encoding ZsGreen1green fluorescent protein, wherein the third nucleotide sequence isshown in SEQ ID NO: 11, an internal ribosome entry site (IRES) ribosomebinding sequence, wherein the IRES ribosome binding sequence is shown inSEQ ID NO: 12; and an eWPRE enhanced marmot hepatitis B viruspost-transcriptional controlling element for enhancing transgenicexpression efficiency, wherein the eWPRE enhanced marmot hepatitis Bvirus post-transcriptional controlling element is shown in SEQ ID NO:13; a sequence of the human EF1αpromoter is shown in SEQ ID NO: 14; thefirst nucleotide sequence of the OCTS chimeric receptor structuraldomain comprises: a fourth nucleotide sequence encoding a cluster ofdifferentiation 8 (CD8) leader chimeric receptor signal peptide, asshown in SEQ ID NO: 15, a fifth nucleotide sequence encoding a firstPDL1 single-chain antibody light chain, as shown in SEQ ID NO: 16, asixth nucleotide sequence encoding a second PDL1 single-chain antibodyheavy chain, as shown in SEQ ID NO: 17, a seventh nucleotide sequenceencoding a first mesothelin single-chain antibody light chain, as shownin SEQ ID NO: 18, an eighth nucleotide sequence encoding a secondmesothelin single-chain antibody heavy chain, as shown in SEQ ID NO: 19,a ninth nucleotide sequence encoding an antibody Inner-Linker, as shownin SEQ ID NO: 20, a tenth nucleotide sequence encoding a single-chainantibody Inter-Linker, as shown in SEQ ID NO: 21, an eleventh nucleotidesequence encoding a CD8 hinge chimeric receptor linker, as shown in SEQID NO: 22, a twelfth nucleotide sequence encoding a CD8 transmembranechimeric receptor transmembrane domain, as shown in SEQ ID NO: 23, athirteenth nucleotide sequence of a chimeric receptor T cell activationdomain of T cell receptor (TCR), as shown in SEQ ID NO: 26, and afourteenth nucleotide sequence of a chimeric receptor co-stimulatordomain; wherein the chimeric receptor co-stimulator domain is at leastone selected from the group consisting of 4-1BB, ICOS, CD27, OX40(CD134), CD28, MYD88, IL1R1, CD70, TNFRSF19L, TNFRSF27, TNFRSF1OD,TNFRSF13B, and TNFRSF18.
 2. The OCTS-based CAR-T vector of claim 1,wherein, the lentiviral packaging cis-element employs asecond-generation lentiviral vector or a third-generation lentiviralvector; wherein the second-generation lentiviral vector comprises: alentiviral 5 terminal long terminal repeat (LTR), as shown in SEQ ID NO:5, a lentiviral 3 terminal Self-Inactivating LTR, as shown in SEQ ID NO:6, a Gag cis-element, as shown in SEQ ID NO: 7, a rev-responsive element(RRE) cis-element, as shown in SEQ ID NO: 8, an env cis-element, asshown in SEQ ID NO: 9, and a cPPT cis-element as shown in SEQ ID NO: 10;the third-generation lentiviral vector comprises: the lentiviral 5terminal LTR, as shown in SEQ ID NO: 5, the lentiviral 3 terminalself-Inactivating LTR, as shown in SEQ ID NO: 6, the Gag cis-element, asshown in SEQ ID NO: 7, the RRE cis-element, as shown in SEQ ID NO: 8,the env cis-element, as shown in SEQ ID NO: 9, the cPPT cis-element, asshown in SEQ ID NO: 10, and a Rous-sarcoma-virus (RSV) promoter, asshown in SEQ ID NO:
 4. 3. The OCTS-based CAR-T vector of claim 1,wherein, the fifth nucleotide sequence encoding the first PDL1single-chain antibody light chain, as shown in SEQ ID NO: 16, the sixthnucleotide sequence encoding the second PDL1 single-chain antibody heavychain, as shown in SEQ ID NO: 17, the seventh nucleotide sequenceencoding the first mesothelin single-chain antibody light chain, asshown in SEQ ID NO: 18, the eighth nucleotide sequence encoding thesecond mesothelin single-chain antibody heavy chain, as shown in SEQ IDNO: 19, the ninth nucleotide sequence encoding the antibodyInner-Linker, as shown in SEQ ID NO: 20, and the tenth nucleotidesequence encoding the single-chain antibody Inter-Linker, as shown inSEQ ID NO: 21, are connected in a sequential way or in a turned way;wherein, in the sequential way, the fifth nucleotide sequence encodingthe first PDL1 single-chain antibody light chain is connected to theseventh nucleotide sequence encoding the first mesothelin single-chainantibody light chain through the tenth nucleotide sequence encoding thesingle-chain antibody Inter-Linker and is connected to the sixthnucleotide sequence encoding the second PDL1 single-chain antibody heavychain-through the ninth nucleotide sequence encoding the antibodyInner-Linker, and the seventh nucleotide sequence encoding the firstmesothelin single-chain antibody light chain is connected to the eighthnucleotide sequence encoding the second mesothelin single-chain antibodyheavy chain through the ninth nucleotide sequence encoding the antibodyInner-Linker; and in the turned way, the seventh nucleotide sequenceencoding the first mesothelin single-chain antibody light chain isconnected to the eighth nucleotide sequence encoding the secondmesothelin single-chain antibody heavy chain through the ninthnucleotide sequence encoding the antibody Inner-Linker, the fifthnucleotide sequence encoding the first PDL1 single-chain antibody lightchain is connected to the eighth nucleotide sequence encoding the secondmesothelin single-chain antibody heavy chain through the tenthnucleotide sequence encoding the single-chain antibody Inter-Linker, andthe sixth nucleotide sequence encoding the second PDL1 single-chainantibody heavy chain is connected to the seventh nucleotide sequenceencoding the first mesothelin single-chain antibody light chain throughthe antibody Inter-Linker.
 4. The OCTS-based CAR-T vector of claim 1,wherein, the PDL1 single-chain antibody is shown in SEQ ID NO:
 27. 5.The OCTS-based CAR-T vector of claim 1, wherein, the eWPRE enhancedmarmot hepatitis B virus post-transcriptional controlling element hassix enhanced nucleotide mutations including: g.396G>A, g.397C>T,g.398T>C, g.399G>A, g.400A>T, and g.411A>T.
 6. The OCTS-based CAR-Tvector of claim 1, wherein, an expression of the OCTS chimeric receptorstructural domain is started by the human EF1αpromoter, and the CD8leader chimeric receptor signal peptide on a N terminal of OCTS codingsequence is configured to guide a location of OCTS protein oncytomembrane; a double antigen recognition domain is formed by the PDL1single-chain antibody light chain, the second PDL1 single-chain antibodyheavy chain, the first mesothelin single-chain antibody light chain andthe second mesothelin single-chain antibody heavy chain for recognizingtarget antigens; the CD8 hinge chimeric receptor linker is configured toanchor scFv on outside of the cytomembrane; the CD8 transmembranechimeric receptor transmembrane domain is configured to fix entirechimeric receptors on the cytomembrane; the CD28 chimeric receptorco-stimulator is configured to stimulate in-vitro T lymphocyteactivation and killing effect on in-vivo tumor cells; the CD134 chimericreceptor co-stimulator is configured to facilitate T lymphocyteproliferation and factor secretion and enhance tumor immunity,contributing to a long-term survival of memory T cells; the TCR chimericreceptor T cell activation domain is configured to activate anexpression of downstream signals; the PDL1 single-chain antibodyefficiently binds to PDL1, blocks negative control signals andclinically suppresses tumor immune evasion, thus improving a curativeeffect of CAR-T in cellular immunotherapy; and when the double antigenrecognition domain is bound to the target antigens, signals aretransmitted into cells through chimeric receptors, thereby creating aseries of biological effects including T cell proliferation, increasedcell factor secretion, increased antiapoptosis secretion, delayed celldeath and target cell lysis.
 7. The OCTS-based CAR-T vector of claim 1,wherein, the chimeric receptor co-stimulator domain is a combination ofCD28 chimeric receptor co-stimulator having a fifteenth nucleotidesequence of SEQ ID NO: 24 and CD134 chimeric receptor co-stimulatorhaving a sixteenth nucleotide sequence of SEQ 1D NO:
 25. 8. TheOCTS-based CAR-T vector of claim 1, wherein, the first PDL1 single-chainantibody light chain, the second PDL1 single-chain antibody heavy chain,the first mesothelin single-chain antibody light chain, the secondmesothelin single-chain antibody heavy chain and the PDL1 single-chainantibody are humanized.
 9. A method for preparing the OCTS-based CAR-Tvector for targeting the malignant tumors expressing the mesothelin, thePDL1, or both of the mesothelin and the PDL1 according to claim 1,comprising the following steps: (1) modifying the lentiviral skeletonplasmid to carry the AmpR sequence containing ampicillin resistance geneas shown in SEQ ID NO: 1, the prokaryotic replicon pUC Ori sequence asshown in SEQ ID NO: 2, the virus replicon SV40 Ori sequence as shown inSEQ ID NO: 3, the lentiviral packaging cis-element for lentiviralpackaging, the third nucleotide sequence encoding the ZsGreen1 greenfluorescent protein as shown in SEQ ID NO: 11, the IRES ribosome bindingsequence as shown in SEQ ID NO: 12, and the eWPRE enhancedpost-transcriptional regulatory element of Groundhog hepatitis B virusas shown in SEQ ID NO: 13; (2) combining the human EF1α promoter asshown in SEQ ID NO: 14, the first nucleotide sequence encoding the OCTSchimeric receptor structural domain and the second nucleotide sequenceencoding the PDL1 single-chain antibody as shown in SEQ ID NO: 27 into adesign scheme for an OCTS chimeric receptor, and cloning the designscheme into the lentiviral skeleton plasmid by digestion, ligation andrecombination to obtain recombinant lentiviral plasmids designed by athird-generation OCTS; (3) transfecting HEK293T/17 cells by therecombinant lentiviral plasmids with lentiviral packaging plasmidspPac-GP and pPac-R and membrane protein pEnv-G respectively; after therecombinant lentiviral plasmids are transcribed and expressed in theHEK293T/17 cells, and recombinant lentiviral vectors packagedsuccessfully are released into cell culture supernatants, collectingsupernatants containing the recombinant lentiviral vectors; and (4)purifying the supernatants by column purification of filtration,adsorption and elution to obtain the recombinant lentiviral vectors,respectively.
 10. The method of claim 9, wherein, in the step (4), thefiltration is performed by controlling a volume of each of thesupernatants from 200 ml to 2000 ml with a vacuum degree from −0.5 MPAto 0.9 MPA to prevent a loss of the recombinant lentiviral vectorscaused by blockage; the adsorption is performed by controlling a pHvalue of a solution from 6 to 8 to prevent the recombinant lentiviralvectors from inactivating due to a change of pH, and the elution isperformed by controlling an ionic strength of an eluent from 0.5 M to1.0 M to prevent a change of the ionic strength leading to incompleteelution or inactivation of the recombinant lentiviral vectors.
 11. TheOCTS-based CAR-T vector of claim 1, wherein, the OCTS-based CAR-T vectoris applied in a preparation of drugs for targeting the malignant tumorsexpressing the mesothelin, the PDL1, or both of the mesothelin and thePDL1.
 12. The method of claim 9, wherein, the lentiviral packagingcis-element employs a second-generation lentiviral vector or athird-generation lentiviral vector; wherein the second-generationlentiviral vector comprises: a lentiviral 5 terminal long terminalrepeat (LTR), as shown in SEQ ID NO: 5, a lentiviral 3 terminalSelf-Inactivating LTR, as shown in SEQ ID NO: 6, a Gag cis-element, asshown in SEQ ID NO: 7, a rev-responsive element (RRE) cis-element, asshown in SEQ ID NO: 8, an env cis-element, as shown in SEQ ID NO: 9, anda cPPT cis-element as shown in SEQ ID NO: 10; the third-generationlentiviral vector comprises: the lentiviral 5 terminal LTR, as shown inSEQ ID NO: 5, the lentiviral 3 terminal self-Inactivating LTR, as shownin SEQ ID NO: 6, the Gag cis-element, as shown in SEQ ID NO: 7, the RREcis-element, as shown in SEQ ID NO: 8, the env cis-element, as shown inSEQ ID NO: 9, the cPPT cis-element, as shown in SEQ ID NO: 10, and aRous-sarcoma-virus (RSV) promoter, as shown in SEQ ID NO:
 4. 13. Themethod of claim 9, wherein, the fifth nucleotide sequence encoding thefirst PDL1 single-chain antibody light chain, as shown in SEQ ID NO: 16,the sixth nucleotide sequence encoding the second PDL1 single-chainantibody heavy chain, as shown in SEQ ID NO: 17, the seventh nucleotidesequence encoding the first mesothelin single-chain antibody lightchain, as shown in SEQ ID NO: 18, the eighth nucleotide sequenceencoding the second mesothelin single-chain antibody heavy chain, asshown in SEQ ID NO: 19, the ninth nucleotide sequence encoding theantibody Inner-Linker, as shown in SEQ ID NO: 20, and the tenthnucleotide sequence encoding the single-chain antibody Inter-Linker, asshown in SEQ ID NO: 21, are connected in a sequential way or in a turnedway; wherein, in the sequential way, the fifth nucleotide sequenceencoding the first PDL1 single-chain antibody light chain is connectedto the seventh nucleotide sequence encoding the first mesothelinsingle-chain antibody light chain through the tenth nucleotide sequenceencoding the single-chain antibody Inter-Linker and is connected to thesixth nucleotide sequence encoding the second PDL1 single-chain antibodyheavy chain through the ninth nucleotide sequence encoding the antibodyInner-Linker, and the seventh nucleotide sequence encoding the firstmesothelin single-chain antibody light chain is connected to the eighthnucleotide sequence encoding the second mesothelin single-chain antibodyheavy chain through the ninth nucleotide sequence encoding the antibodyInner-Linker; and in the turned way, the seventh nucleotide sequenceencoding the first mesothelin single-chain antibody light chain isconnected to the eighth nucleotide sequence encoding the secondmesothelin single-chain antibody heavy chain through the ninthnucleotide sequence encoding the antibody Inner-Linker, the fifthnucleotide sequence encoding the first PDL1 single-chain antibody lightchain is connected to the eighth nucleotide sequence encoding the secondmesothelin single-chain antibody heavy chain through the tenthnucleotide sequence encoding the single-chain antibody Inter-Linker, andthe sixth nucleotide sequence encoding the second PDL1 single-chainantibody heavy chain is connected to the seventh nucleotide sequenceencoding the first mesothelin single-chain antibody light chain throughthe antibody Inter-Linker.
 14. The method of claim 9, wherein, the PDL1single-chain antibody is shown in SEQ ID NO:
 27. 15. The method of claim9, wherein, the eWPRE enhanced marmot hepatitis B viruspost-transcriptional controlling element has six enhanced nucleotidemutations including: g.396G>A, g.397C>T, g.398T>C, g.399G>A, g.400A>T,and g.411A>T.
 16. The method of claim 9, wherein, an expression of theOCTS chimeric receptor structural domain is started by the human EF1αpromoter, and the CD8 leader chimeric receptor signal peptide on a Nterminal of OCTS coding sequence is configured to guide a location ofOCTS protein on cytomembrane; a double antigen recognition domain isformed by the PDL1 single-chain antibody light chain, the second PDL1single-chain antibody heavy chain, the first mesothelin single-chainantibody light chain and the second mesothelin single-chain antibodyheavy chain for recognizing target antigens; the CD8 hinge chimericreceptor linker is configured to anchor scFv on outside of thecytomembrane; the CD8 transmembrane chimeric receptor transmembranedomain is configured to fix entire chimeric receptors on thecytomembrane; the CD28 chimeric receptor co-stimulator is configured tostimulate in-vitro T lymphocyte activation and killing effect on in-vivotumor cells; the CD134 chimeric receptor co-stimulator is configured tofacilitate T lymphocyte proliferation and factor secretion and enhancetumor immunity, contributing to a long-term survival of memory T cells;the TCR chimeric receptor T cell activation domain is configured toactivate an expression of downstream signals; the PDL1 single-chainantibody efficiently binds to PDL1, blocks negative control signals andclinically suppresses tumor immune evasion, thus improving a curativeeffect of CAR-T in cellular immunotherapy; and when the double antigenrecognition domain is bound to the target antigens, signals aretransmitted into cells through chimeric receptors, thereby creating aseries of biological effects including T cell proliferation, increasedcell factor secretion, increased antiapoptosis secretion, delayed celldeath and target cell lysis.
 17. The method of claim 9, wherein, thechimeric receptor co-stimulator domain is a combination of CD28 chimericreceptor co-stimulator having a fifteenth nucleotide sequence of SEQ IDNO: 24 and CD134 chimeric receptor co-stimulator having a sixteenthnucleotide sequence of SEQ ID NO:
 25. 18. The method of claim 9,wherein, the first PDL1 single-chain antibody light chain, the secondPDL1 single-chain antibody heavy chain, the first mesothelinsingle-chain antibody light chain, the second mesothelin single-chainantibody heavy chain and the PDL1 single-chain antibody are humanized.19. The OCTS-based CAR-T vector of claim 2, wherein, the OCTS-basedCAR-T vector is applied in a preparation of drugs for targeting themalignant tumors expressing the mesothelin, the PDL1, or both of themesothelin and the PDL1.
 20. The OCTS-based CAR-T vector of claim 3,wherein, the OCTS-based CAR-T vector is applied in a preparation ofdrugs for targeting the malignant tumors expressing the mesothelin, thePDL1, or both of the mesothelin and the PDL1.